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Dengue type 2 virus (DV) induces the generation of T helper cells (TH) in the mouse spleen. These TH on adoptive transfer to DV primed syngeneic mice enhance the clonal expansion of antigen-specific antibody plaque forming cells. The present study was undertaken to investigate the role of M phi and Ca2+ in the induction of DV-specific TH. The results show that M phi are obligatory for the induction of DV-specific TH. It was observed that DV antigen stimulate the influx of calcium ion (Ca2+) into T cells as shown by radiolabelled (45Ca) and the influx of Ca2+ increases with time of incubation, reaching a maximum level after 1 hr of incubation. The proliferation of T cells was found to be dependent on presence of Ca2+ as it was inhibited by Ca channel blocking drugs and in the absence of Ca in the medium. Thus showing that Ca2+ plays an important role in the induction of DV-specific TH cells.

During dengue type 2 virus (DV) infection of mice a unique cytokine, the cytotoxic factor (CF), is produced which reproduces the pathological lesions seen in patients of dengue haemorrhagic fever (DHF). Recently we have observed a CF-like protein in the sera of DHF cases. The present study was undertaken to investigate whether DV can stimulate human peripheral blood mononuclear cells (PBMC) in vitro to produce human CF (hCF). Cultures prepared from PBMC or its enriched subpopulations were inoculated with 1000 LD50 of DV and controls with normal mouse brain suspension (NMB). Aliquots of cultures were harvested daily from 24 h to 96 h and their supernatant (CS) and cells were separated. CS were screened for viral titres and for the presence of hCF by cytotoxicity assay, inhibition ELISA, dot blot and Western blot tests using anti-mouse-CF antibodies. The RNA from the cells was screened in Northern blot and dot blot tests for the presence of mRNA for CF. It was observed that hCF appeared in the CS of DV-infected culture of PBMC and T-enriched cells at 48 h and was present until 96 h; no CF was detected in CS of B cells or monocyte cultures. The production of hCF was abrogated by pretreatment of the T cells with anti-CD4 antibodies but not with anti-CD8 antibodies, indicating that hCF was produced by CD4+ T cells. The Northern blot analysis using oligonucleotide probes prepared on the basis of amino-terminal sequence of mouse CF, showed presence of mRNA for hCF in PBMC and T cell cultures. DV replicated in PBMC cultures with peak titres at 48 h. The findings of the present study demonstrate that DV-induced hCF is produced by human T cells.

The present study was undertaken to investigate the role of calcium in the production of dengue type 2 virus (DV) induced suppressor cytokine (SF) and in transmission of the suppressor signal via syngeneic macrophage (M phi) to recruit the second subpopulation of suppressor T(TS2) cell. Effect of calcium channel blocking drugs, Verapamil and Nifedipine, on the production and activity of SF was investigated. The production of SF was inhibited in a dose dependent manner by the calcium channel blockers. The suppressor activity of SF was also inhibited by calcium channel blocking drugs. SF could not be produced when spleen cells were cultured in a calcium free medium, the production being restored on substitution with calcium chloride. Treatment of M phi with the calcium channel blockers inhibited the transmission of the suppressor signal from TS1 to TS2 cells in a dose dependent manner. The influx of calcium during transmission of the suppressor signal was studied by measuring the 45Ca uptake. Calcium channel blocking drugs inhibited the 45Ca uptake by T lymphocytes. Thus, presence of calcium is obligatory for the production and suppressor activity of SF and it plays a critical role in transmission of the suppressor signal.

In this study, viruria following Japanese encephalitis virus (JEV) infection in mice has been shown to appear earlier in pregnant than in normal mice with proteinuria and haematuria. This was related to the production of splenic macrophage derived neutrophil chemotactic factor (MDF) following JEV infection. Intravenous inoculation of MDF in mice resulted in leakage of cells, proteins and erythrocytes in the urine as a result of altered capillary permeability. The isolation of virus from kidney did not correlate with the shedding of virus in the urine. The histological examination of sections of kidneys showed no morphological damage; however, ultrastructural degenerative changes in the mesangial cells were observed following JEV infection. These data suggest that JEV-induced macrophage derived factor regulates the leakage of proteins, erythrocytes and cells into the urine.

The vascular effects of Japanese encephalitis virus (JEV)-stimulated splenic macrophage-derived neutrophil chemotactic factor (MDF) were evaluated in mice. Intraperitoneal injection of MDF in mice resulted in a rapid increase in capillary permeability in a dose-dependent manner as assessed by leakage of intravenously injected radiolabelled albumin ([125I]-albumin) or Evans blue dye. Intradermal inoculation of MDF in rabbits caused [51Cr]-labelled neutrophil emigration and accumulation into injected sites. Peak plasma leakage and neutrophil infiltration were observed at 1 h following MDF inoculation, and plasma leakage was restored by 2.5 h. The increase in capillary permeability was sensitive to pretreatment of mice with avil and ranitidine (H1 and H2 histamine receptor blockers, respectively), resulting in abrogation of the response; indomethacin, a prostaglandin synthetase inhibitor, did not have any effect.

Dengue type 2 virus (DV)-induced cytotoxic factor (CF) is capable of reproducing various pathological lesions in mice that are seen in human dengue. The present study was undertaken to investigate the protective effect of active immunization of mice with CF. Mice were immunized with 5 microgram of CF and prevention of CF-induced increase in capillary permeability and damage to the blood-brain barrier were studied at weekly intervals, up to 48 weeks, by challenging with 3 microgram of CF. Maximum protection against increase in capillary permeability and damage to the blood-brain barrier was observed in week 4 after immunization. A breakthrough in the protection occurred with higher doses of CF in a dose-dependent manner. Challenge with a lethal intracerebral (i.c.) dose of DV showed significantly prolonged mean survival time and delayed onset of symptoms of sickness in the immunized mice compared with the normal mice, but the titre of the virus in the brain was similar in the two groups. On i.p. challenge with the virus the protection against damage to the blood-brain barrier was 86 +/- 7% at week 4 and 17 +/- 4% at week 26 after immunization. Sera obtained from the immunized mice showed the presence of CF-specific antibodies by ELISA, Western blot, and by neutralization of the cytotoxic activity of CF in vitro. The present study describes successful prevention of a cytokine-induced pathology by specific active immunization.

The role of cytosolic Ca2+ concentration in neutrophils stimulated with macrophage derived neutrophil chemotactic factor (MDF) produced following Japanese encephalitis virus (JEV) infection in mice was correlated with cell functions. MDF-induced Ca2+ influx from extracellular milieu and release from intracellular store resulted in rise of cytosolic Ca2+ in a dose-dependent manner and was independent of protein kinase C. Macrophages and B cells did not show cytosolic Ca2+ changes while T lymphocytes showed slight rise when stimulated with MDF. Neutrophil chemotaxis in the absence of Ca2+ was slightly different from that in presence of Ca2+. Pretreatment of neutrophils with 3,4,5-trimethoxy-benzoic acid-8-(diethylamine)-octylester (TMB-8) inhibited the chemotaxis. It was observed that superoxide production and degranulation by neutrophils after stimulation with MDF was not dependent on the presence of extracellular calcium, but stripping of intracellular calcium resulted in abrogation of neutrophil activation. Thus, mobilization of intracellular calcium seems to be necessary for neutrophil activation by MDF.

The present study was undertaken to investigate the presence of receptors for the dengue virus-induced suppressor cytokine (SF) on mouse macrophages (Mphi), T and B lymphocytes. It was observed that 125I-SF bound to T and B cells to a variable degree through both high and low affinity receptors. The numbers of high affinity receptors on T and B cells were 35,000 and 16,000/cell respectively and those of low affinity receptors were 0.72 x 10(6) and 0.33 x 10(6)/cell respectively. The binding of 125I-SF was also shown by removal of the biological activity following adsorption on Mphi monolayers. The suppressor activity was inhibited by pretreatment of SF with anti-SF-antisera. The binding of 125I-SF to Mphi was saturable, reversible and dependent on pH, time and temperature. This shows the presence of a specific receptor for SF on mouse Mphi, T and B lymphocytes.

The effect of dengue type 2 virus (DV) and the cytokines induced by it, on the chemiluminescence (CL) response of mouse spleen cells was studied by luminol-dependent CL. Following intracerebral (i.c.) inoculation of DV, an initial increase in CL response was observed which was followed by a sharp decline from the 4th day post-infection with lowest values on days 5 and 6. DV-induced depression of CL was inhibited by pretreatment of mice with antisera against the two DV-induced cytokines, the cytotoxic factor (CF) and the cytotoxin (CF2), but antisera against the other two helper (HF) or suppressor (SF) cytokines had minimal effect. Inoculation of CF or CF2 into mice depressed the CL in dose-dependent manner at 2 hours but increased it at the 3rd day. Inoculation of HF or SF had no significant effect. Treatment of mice with the cytokines in presence of the specific antisera had an enhancing effect on the CL response. The antisera alone had no effect on CL. Thus, these findings indicate that DV infection impaired the generation of respiratory burst, and the impairment appeared to be mediated by two DV-induced cytokines (CF/CF2). Targetting of the cytokines to M phi was significantly enhanced in the presence of specific anti-cytokine antibodies.

The effects of Japanese encephalitis virus (JEV) and JEV-induced macrophage derived neutrophil chemotactic factor (MDF) on respiratory burst and granule exocytosis in neutrophils were studied and were compared with N-formyl-methionyl-leucyl-phenylalanine (FMLP). JEV-stimulated neutrophils released reactive oxygen metabolites with maximum activity between days 7 and 9. The response in mice was sensitive to anti-JEV-antisera treatment. Stimulation by MDF resulted in quick release of superoxide and granule enzymes from neutrophils upon both in-vivo and in-vitro stimulation in a dose dependent manner. The effect was abrogated by the MDF-specific antisera treatment. These responses were similar in kinetics and magnitude to those produced in response to FMLP. The respiratory burst elicited by MDF was abrogated by staurosporine, indicating that neutrophil activation and signal transduction by MDF and FMLP are dependent on protein kinase C.

Dengue type 2 virus (DV)-induced suppressor cytokine (SF) is composed of two polypeptide chains (alpha and beta) and has a receptor on macrophages (M phi), which has been purified. The present study was undertaken to study the binding of the two polypeptide chains of SF to M phi and its purified receptor (SF-R) protein. It was observed that both alpha and beta-chains of SF, purified by high performance liquid chromatography (HPLC), bound to M phi cell, but only alpha-chain bound to SF-R protein. Only H-2A positive M phi bound SF. Anti-H-2Ak mAb blocked the binding of SF to M phi but had no effect on binding of SF to SF-R. Both anti-SF-antiserum and anti-SF-R-antiserum blocked the binding of SF to M phi. The binding of the beta-chain of SF to M phi was blocked by anti-H-2Ak mAb while the anti-SF-R-antiserum blocked the binding of alpha-chain on M phi. Thus, the alpha-chain of SF binds to SF-R and the beta-chain binds to H-2A determinants on M phi.

Macrophage (M phi) process dengue type 2 virus (DV) antigen and present it to B cells leading to their clonal expansion and transmit DV-specific helper cytokine (HF) signal to B cells. The present study was, therefore, undertaken to investigate the binding of DV or HF-pulsed M phi with 51Cr- labelled B lymphocytes. The binding was dependent on the number of DV antigen positive M phi or dose of HF, period (peak with DV at 24 h and with HF at 4 h) and temperature of incubation. The binding was significantly increased by treating DV-pulsed M phi with DV-specific helper T cells or HF. Glutaraldehyde fixed M phi (which do not process DV antigen (Rizvi et al. 1989)) had poor binding capacity. The binding was DV- or HF-specific as it was inhibited by treatment of M phi with specific antisera. The findings thus show that M phi and B lymphocytes are actively bridged via DV antigen and HF to effectively transmit the antigen-specific signal.

Dengue type 2 virus (DV)-induced suppressor cytokine (SF) binds to macrophages to transmit the suppressor signal to recruit the second subpopulation of suppressor T cells. The present study was undertaken to identify and purify the receptor for SF (SF-R) on macrophages. The binding of 125I-SF to macrophages was saturable and reversible. Scatchard analysis showed the presence of both high (54,000/cell) and low (1.78 x 10(6)/cell) affinity receptor sites. The binding of 125I-SF to macrophages was inhibited by pretreatment of macrophages with anti-SF antiserum but not by a heterologous antiserum. Normal mouse peritoneal macrophage membrane was solubilized with Triton-X-100 and the components separated by low pressure liquid chromatography (LPLC) to purify SF-R. The presence of SF binding moiety (SF-R) was screened at each step of purification. The purified SF-R resolved into two bands of 45-50 kD mol. wt on SDS-PAGE. 125I-SF+SF-R complex run on SDS-PAGE showed a single band at about 55-60 kD mol. wt by autoradiography. Anti-SF-R antiserum reacted with SF-R in a Western blot test; the reaction was abolished by pretreatment of the blots with proteinase K, but not by pretreatment with periodic acid. SF-R was composed of two polypeptide chains (alpha and beta) which were obtained in pure form by high performance liquid chromatography (HPLC) of dithiothreitol- and iodoacetamide-treated SF-R. Only the beta chain bound SF.

The helper T cells (TH) generated in dengue type 2 virus (DV) infection of mice produce a soluble helper cytokine (HF) which enhances the clonal expansion of DV-specific IgM antibody plaque forming cells (PFC). The present study was undertaken to investigate the mechanism of transmission of the helper signal from TH and HF to B cells. It was observed that TH could transmit the helper signal to B cells by direct cell to cell contact, but HF could not do so without the presence of live macrophages (M phi). HF was adsorbed by both heat killed and live M phi but the former could not transmit it to B cells. Both the polypeptide chains of HF bind to M phi. HF remains on the surface of M phi and can be retrieved completely by contact with B cells for 40 min. The helper signal from TH or HF-adsorbed M phi could not be transmitted to B cells when they were separated from each other by a cell impermeable membrane. The enhancement of PFC count is greater when the signal is transmitted by HF-adsorbed M phi as compared to that by TH alone. Thus, even with lower frequency of TH a significant number of B cells may be triggered with the help of HF and M phi. The findings thus show that the DV-specific helper signal could be transmitted only by a close physical contact of the plasma membranes of the signal presenting cells (TH or HF-adsorbed M phi) and B cells.

In this study we have shown, for the first time, that Japanese encephalitis virus (JEV) and a low molecular weight (10 kDa) macrophage-derived neutrophil chemotactic factor (MDF) produced following JEV infection in mice could cause an alteration in the permeability of the blood-brain barrier resulting in leakage of plasma protein bound Evans blue dye and radiolabelled erythrocytes in brain. The maximum leakage occurred at day 6 after intracerebral (i.c.) JEV infection and was sensitive to anti-JEV antisera. Further, MDF caused peak leakage of dye and radiolabelled erythrocytes at 1 h post inoculation with a decline thereafter. Complete restoration of the integrity of the blood-brain barrier occurred by the 4th hour. The extent of leakage was dose dependent and showed a direct correlation between the level of MDF, clinical sickness and virus titre in brain. Anti-MDF antisera protected the mice against the effects of MDF. These findings show that JEV-induced cytokine, MDF, alters the integrity of the blood-brain barrier and thus controls the cellular and plasma leakage into the CNS.

Dengue type 2 virus (DV) induces a subpopulation of T lymphocytes of mouse spleen to secrete a soluble helper cytokine (HF) which enhances the DV-specific IgM antibody plaque forming cells (PFC). The present study undertaken to purify and characterize HF shows that it can be purified by low pressure liquid chromatography (LPLC) using Sephacryl S-200 column. HF consisted of two subunits, having a M(r) of 65-68 kDa on SDS-PAGE, and both had similar activity. The isoelectric point of HF was 6.5. HF-specific antisera (HFAS) raised in mice neutralized the activity of HF in mice, reacted with it in a Western blot assay, and bound HF in an immunosorbent column. HF bound to DV-antigen in an immunosorbent column and enhanced only the DV-specific PFC. HF had no effect on PFC against heterologous antigens such as Japanese encephalitis virus, Coxsackie B4 virus or sheep red blood cells. HF generated in mice of H-2k haplotype, enhanced DV-specific PFC in the same strain of mice but had no effect on that in the H-2d or H-2q haplotype strains of mice. Thus, DV-induced HF with a M(r) of 65-68 kDa, antigen-specificity and genetic-restriction differs from most of the similarly acting cytokines but appears similar to the cell-free form of T cell receptor alpha beta dimer.

The present study was undertaken to purify the dengue type 2 virus (DV)-induced cytotoxic factor (CF) and analyse its amino-terminal sequence. Spleens collected from DV-infected moribund mice were made into a single cell suspension and cultured for 24 h. The culture supernatant was purified using fast protein liquid chromatography (FPLC) and polyacrylamide gel electrophoresis (PAGE). CF could be purified in a single step by FPLC and native PAGE. The isoelectric point of CF was pH 6.5. Purified CF had a molecular weight of 45 kDa on native-PAGE while on SDS-PAGE it dissociated into two bands of 20 and 25 kDa. Anti-CF-antisera reacted specifically with CF bands separated on native and SDS-PAGE in a Western blot assay. A sequence of 19 amino-acids of the N-terminus of CF was analysed which on comparison with that of other known cytotoxic proteins indicated that CF differs from them. Thus, CF appears to be a unique cytotoxic protein induced by a virus.

A study was undertaken to determine the adverse foetal outcome in antichlamydial IgM positive asymptomatic pregnant females. An indirect immunoperoxidase assay was done to detect IgM in 78 apparently normal asymptomatic pregnant women during the third trimester and follow up was done till delivery to study the effect of chlamydial infection on foetal outcome. A total of 28 (35.9%) women were positive for antichlamydial IgM while only 3.33 per cent asymptomatic non-pregnant normal women (controls) were positive. 28.7 per cent IgM positive mothers delivered low birth weight (LBW) babies (P less than 0.05), 9.7 per cent had premature labour (PTL) and 4.7 per cent had intrauterine death (IUD). The findings are significant as none of the IgM negative mothers had PTL and IUD and only 2.6 per cent had LBW babies.

Dengue type 2 virus (DV) induces generation of a T cell helper cytokine (HF) in mouse spleen which enhances the antigen-specific antibody plaque forming cell count in syngeneic mice. The present study was undertaken to investigate the molecular structure of HF. It was observed that the activity of HF was abrogated by treatment with reducing agents such as glutathione, ouabain or dithiothreitol (DTT) which cleave disulphide bonds, separating the polypeptide chains. The two polypeptide chains could be purified by high performance liquid chromatography of DTT treated HF. The individual chains had no helper activity, but it could be restored by mixing the two. One chain of the HF bonded to the DV-antigen coupled immunosorbent column and the other to the anti-I-Ak antibody coupled column. Thus, DV-induced HF is a disulphide bonded double chain structure, one chain having antigen and the other having I-A determinants; the presence of both chains is essential for helper activity.

Antibiotic susceptibility testing against 17 antibiotics was done on 96 strains of various species of coagulase negative staphylococci by Stokes method. Hundred per cent sensitivity was found against vancomycin and cefotaxime and about 90 per cent against ciprofloxacin, clavulanate potentiated amoxycillin, cloxacillin and clindamycin. Strains showed highest resistance against cotrimoxazole (77.08%) and tetracycline (64.59%). Clavulanate potentiated amoxycillin was found to be highly active against penicillin, ampicillin and amoxycillin resistant organisms. The results highlight the importance of antibiotic resistance typing among coagulase negative staphylococci species which are increasingly being reported from serious clinical infections making empiric therapy and selection of antibiotics difficult in these infections.

The mechanism of neutrophil leucocytosis in cases of Japanese encephalitis is not known. We here report that during Japanese encephalitis virus (JEV) infection in mice the splenic macrophages secrete a chemotactic factor that attracts the neutrophils. The peak activity of macrophage derived factor (MDF) was observed on day 7 following infection. The MDF acted in a dose-dependent manner. This chemoattractant was purified by low pressure liquid chromatography and gave a single band of 10 kD on silver stained polyacrylamide gel. The MDF was found to be heat resistant and sensitive to prolonged incubation with proteases.

From Sept. 1986 to Jan. 1989, a hospital-based study was conducted on 736 children, under 5 years of age, with acute respiratory infection. Nasopharyngeal secretions were examined for viruses by culture and by immunofluorescence. Viruses were detected in 22% of specimens: respiratory syncytial (5%), parainfluenza (5%), influenza A (4%), influenza B (2%), adenovirus (3%), measles (3%). The highest rates of detection were with patients diagnosed clinically as pneumonia or upper respiratory tract infection. The case fatality rate was very high (43%) in children with measles virus infection.

In a study group of 40 children who had been admitted to hospital with acute encephalitis, the disease was due to infection with Japanese encephalitis virus (JEV). Three children developed recurrence of disease 8-9 months later. No virus had been isolated from these three patients during the acute stage of their illness, but virus was recovered from all during the recurrence phase by co-cultivation of their peripheral blood mononuclear cells in primary mouse embryo fibroblast cultures. Virus was also recovered by co-cultivation of peripheral blood mononuclear cells collected 8 months after their acute disease from three out of eight randomly selected asymptomatic children within the study group but not from similar cultures set up from JEV-seronegative children used as controls. Virus was also isolated by co-cultivation of T lymphocytes of asymptomatic children as detected by indirect immunofluorescence or by inoculation in mice.

A breakdown of the blood-brain barrier occurred in mice inoculated intracerebrally (i.c.) or intraperitoneally (i.p.) with dengue virus type 2 (DEN2). This resulted in leakage of protein-bound Evans blue dye and 51Cr-labelled erythrocytes into the brain tissue. The leakage increased with time after infection and coincided with an increase of a DEN2-induced cytokine, the cytotoxic factor (CF), in the spleens of such mice. The titres of virus in the brain increased exponentially in i.c. inoculated mice but the virus was not detected in brains of mice given DEN2 by the i.p. route. Similar breakdown of the blood-brain barrier also occurred in mice inoculated intravenously with CF; the damage was dose-dependent and the vascular integrity was restored during the 3 h period after inoculation. Treatment of mice with antihistamine drugs, blocking H1 or H2 receptors, decreased the DEN2-induced protein leakage by up to 50% in i.c. inoculated mice and up to 92% in those inoculated i.p. Indomethacin, a prostaglandin synthetase inhibitor, had no effect. In i.c. inoculated mice protein leakage was inhibited by about 60% by treatment with CF-specific (CFA) or DEN2-specific antisera (DEN2A) whereas protection was complete with the combined treatment with both antisera. On the other hand, in i.p. inoculated mice the inhibition of protein leakage was 80 to 89% with CFA. These findings show a breakdown of the blood-brain barrier leading to cerebral oedema during DEN2 infection which is mediated via the release of histamine by a virus-induced cytokine.

Forty-one children with acute laboratory confirmed Japanese encephalitis were studied. Serum iron concentrations were consistently low following Japanese encephalitis virus infection, with the levels being of prognostic significance.

The role of calcium ions (Ca2+) in the cytotoxic activity of the dengue type 2 virus (DV)-induced macrophage (M phi) cytotoxin (CF2) was investigated in the present study. The findings show that CF2 prepared in Ca(2+)-free medium had no cytotoxic activity on normal mouse spleen cells suspended in Ca(2+)-free medium but killed the cells suspended in the medium with Ca2+. Substitution with calcium chloride restored the cytotoxic activity of CF2 the optimal dose being 10(-4) M concentration. CF2 induced an influx of Ca2+, as assayed by uptake of radiolabelled calcium chloride (45Ca), in the susceptible target cells, viz. M phi and T lymphocytes. The cytotoxic activity of CF2 as well as the CF2-induced influx of 45Ca was inhibited by treatment of the target cell with the calcium channel blocking drugs verapamil and nifedipine. Thus, the presence of Ca2+ is obligatory for the cytotoxic activity of CF2 and cell death is associated with increased intracellular Ca2+.

Depression of serum iron following Japanese encephalitis virus (JEV) infection was observed in mice. The hypoferraemia was associated with the accumulation of iron in reticulo-endothelial cells in the spleen. Splenectomy (compared with sham-operation) prevented the depression in serum iron concentration after JEV infection. It also prevented the rise in levels of liver iron. The effect of JEV-stimulated, splenic macrophage-derived factor (MDF) was evaluated in causing hypoferraemia. MDF produced a rapid reduction in the serum iron levels with accumulation of iron in spleen. These observations suggest that MDF plays a key role in the regulation of iron metabolism during JEV infection.

It has been shown that macrophages (M phi) process dengue type 2 virus (DV) antigen and present it to B cells leading to their clonal expansion as shown by DV-specific IgM antibody plaque-forming cell (PFC) count in spleen. The present study was undertaken to find out the nature of enzymes responsible for the processing of DV antigen in M phi. DV-pulsed M phi were treated with seven different protease inhibitors and then assayed for antigen presentation to B cells. It was observed that maximum inhibition occurred by treatment of M phi with PMSF, a serine-protease inhibitor. The effect of PMSF was dose dependent and was abolished by using predigested antigen. PMSF inhibited presentation of DV and sheep RBC antigens but had no effect on presentation of bovine serum albumin which does not require processing. The results thus identify the serine group of proteases as the main enzymes involved in processing the DV antigen in M phi.

The present study was undertaken to investigate the role of calcium ions (Ca2+) in the cytotoxic activity of the cytotoxic factor (CF) produced by T lymphocytes of the dengue type 2 virus (DV)-infected mouse spleen. It was observed that CF prepared in Ca2(+)-free medium had no cytotoxic activity on normal mouse spleen cells suspended in Ca2(+)-free medium but had activity on cells suspended in medium having Ca2+. The cytotoxic activity of CF was restored by substitution with calcium chloride, the optimal dose being 10(-7) M. CF induced influx of Ca2+ as measured by uptake of radiolabelled calcium chloride (45Ca), in the susceptible target cells, macrophages (M phi) and T lymphocytes, but had no effect on CF-resistant B lymphocytes. Calcium channel blocking drugs, like verapamil, nifedipine and diltiazem, inhibited the cytotoxic activity of CF and also the CF-induced influx of 45Ca in M phi and T cells. Thus, presence of Ca2+ is obligatory for the cytotoxic activity of CF and the cell death is associated with increased intracellular Ca2+.

Macrophages (M phi) pulsed with dengue type 2 (DV) and Coxsackie B4 (CoxB) viruses present antigen to B lymphocytes leading to their clonal expansion as detected by counting antigen-specific IgM antibody plaque-forming cells (PFC). The present study was undertaken to investigate the site for competition in M phi between the two heterologous antigens, DV and CoxB, for their presentation to B cells. It was observed that DV-pulsed M phi presented antigen to B cells in mice depleted of T cells by treatment with anti-Thy 1.2 monoclonal antibodies. The B cells could not be stimulated in absence of M phi in mice treated with silica. The PFC counts for both the antigens were inhibited when M phi were pulsed simultaneously with DV and CoxB. PFC counts were increased by 53-120% by predigesting the antigens by trypsin. Inhibition of DV-specific response by CoxB was abrogated by predigesting CoxB. A marked reduction in DV-specific PFC response was observed when CoxB was superimposed on M phi pulsed with DV 24 h earlier. CoxB-specific PFC counts were not affected by superimposing DV on M phi pulsed with CoxB 24 h earlier. PFC response to the antigen given to M phi before glutaraldehyde fixation was not affected while that for the antigen given to glutaraldehyde-fixed M phi was markedly depressed. It is concluded that the competition between DV and CoxB for antigen presentation to B cells occurs in M phi at the level of antigen processing.

A total of 740 consecutive children aged between 6 months and 12 years who presented with acute encephalopathic illnesses during a three year period were assessed both clinically and by laboratory investigations. Cerebrospinal fluid was examined for the presence of cells or other abnormal substances, and any organisms were cultured. Blood examination included white cell count and estimations of haemoglobin, urea, glucose, and electrolyte concentrations and serum alanine aminotransferase and aspartate aminotransferase. A firm diagnosis was established in 278 patients (38%). Pyogenic meningitis (n = 134), measles encephalopathy (n = 38), and electrolyte imbalance (n = 23) were important causes in this group, cerebral malaria (n = 4) was uncommon and there were no cases of Reye's syndrome. The diagnoses of the remaining 462 were combined under the heading 'acute unexplained encephalopathy'. Altogether 394 of the 462 patients underwent virological investigations for arboviruses and 92 (23%) had one or more indicators of Japanese encephalitis. No other arboviruses could be isolated. Throat swabs from 187 patients with acute unexplained encephalopathy were studied on monkey kidney tissue cell lines of which 14 were positive (8%). These were identified as adenovirus, parainfluenza, influenza, poliomyelitis, Coxsackie, and echovirus; in two cases the virus was untypable. Japanese encephalitis is an important cause of acute childhood encephalopathy in this region. Clinical features of the illness may be mimicked by several disorders which require specific treatment. Thirty four of the 92 died (37%).

Patterns of serum and C.S.F. IgG, IgA and IgM, and haemagglutination inhibiting (HI) antibodies and complement fixing (CF) antibodies against rubella and cytomegaloviruses respectively and total CSF protein were investigated in 30 depressives, 20 each in neurological and surgical patients. Significant changes in total CSF protein and immunoglobulins in depressives and neurological patients in comparison to surgical subjects indicate an etiological similarity between the two groups. Failure to detect antibodies in C.S.F. of these subjects and statistically insignificant seropositivity refute the claim of viral hypothesis for depression but similar alterations in these body proteins in depressives and neurological patients raise other aetiological possibilities.

This communication reports the clinical features of 92 children with Japanese encephalitis diagnosed on the basis of viral isolation from brain or cerebrospinal fluid and/or a 4-fold or higher rise in antibody titre in paired sera by haemagglutination inhibition (HI) test, followed by detection of specific IgM HI antibody. More than a third of the patients (38%) were from urban areas. A high incidence of fever (94.5%), coma (100%) and convulsions (84.7%) was seen. Focal neurological deficit was found in 29.3 per cent of patients and pleocytosis in the cerebrospinal fluid in 32.3 per cent. An extrapyramidal syndrome developed in 21.3 per cent patients during the convalescent stage. Thirty four (36.9%) patients died while in hospital. Mortality was significantly related to a short prodromal stage, deep coma, abnormalities in tone and breathing and decerebrate posturing. Japanese encephalitis represents a severe disease in children with a high early mortality.

Following Japanese encephalitis virus (JEV) infection in mice the serum iron concentration decreased; the decline started from day 3 and persisted up to day 9 postinfection. No significant difference was found in the total iron-binding capacity of serum in JEV-infected mice compared with that of controls. The hypoferraemia was associated with accumulation of iron in the spleen. Large amounts of stainable iron were demonstrated in splenic macrophages from day 5. This was accompanied by transient anaemia. Thus, the hypoferraemia, following intraperitoneal inoculation of JEV, was attributed to a block in the release of iron from the RES.

A total of 568 children suffering from acute respiratory infections were studied from September, 1986 to June, 1988 using indirect immunofluorescence technique to know the frequency of infections caused by parainfluenza viruses 1 and 3. Though sporadic cases of parainfluenza 1 (2.5%) and parainfluenza 3 (3.2%) were seen round the year, and increase in cases of parainfluenza virus type 1 infection was observed only in October, 1987 in Lucknow.

The mechanism of increased capillary permeability, seen in cases of dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS), is not known. Dengue type 2 virus (DV) is known to induce production of a lymphokine, the cytotoxic factor (CF), by the T lymphocytes of mouse spleen. The data presented here show that intraperitoneal inoculation of CF in mice results in increased capillary permeability in a dose-dependent manner, as shown by leakage of intravenously injected radiolabelled iodine (125I) or Evans blue dye. Peak leakage occurred 30 min after inoculation of CF and the vascular integrity was restored by 2 hr. The increase in capillary permeability was abrogated by pretreatment of mice with anti-CF antibodies, avil (H1 receptor blocker) or ranitidine (H2 receptor blocker). The findings thus show that a DV-induced lymphokine, the CF, increases the capillary permeability via release of histamine.

Capillary permeability is increased in cases of dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS) but its genesis is not known. Dengue type 2 virus (DV) induces production of a cytokine (CF2) by mouse macrophages. The present study was undertaken to investigate the effect of CF2 on capillary permeability. It was observed that intraperitoneal inoculation of CF2 in mice increased the capillary permeability in a dose-dependent manner, as shown by leakage of intravenously injected radioactive iodine (125I) or Evan's blue dye in the peritoneal cavity. Peak leakage occurred at 30 min and the vascular integrity was restored by 1-2 h. The increase in capillary permeability was abrogated by pretreatment of mice with avil (H1 receptor blocker) but not by ranitidine (H2 receptor blocker). The findings thus show that DV-induced CF2 increases the capillary permeability via release of histamine.

Two hundred and thirty children clinically diagnosed as suffering from acute respiratory infection were tested for four major groups of viral aetiological agents, i.e. influenza para-influenza, respiratory syncytial virus (RSV) and adenoviruses using indirect immunofluorescence technique. At least one of the respiratory viruses was identified in 51 (22 per cent) specimens, which included influenza A in 6 (3 per cent), influenza B in 3 (1 per cent), para-influenza type 1 in 3 (1 per cent), para-influenza type 3 in 13 (6 per cent), RSV in 11 (5 per cent) adenovirus in 12 (5 per cent), and dual virus infections in 3 (1 per cent) cases. Maximum number of virus identification was noted in children below 1 year of age, particularly infection with RSV followed by para-influenza and adenoviruses. Value of rapid diagnosis by indirect immunofluorescence technique is stressed.

An indirect immunofluorescent technique for rapid diagnosis of patients of Japanese encephalitis (JE) was developed by demonstrating the viral antigen in CSF cells. The CSF samples of 31 consecutive patients admitted with acute viral encephalitis during 1987 were studied. In 15 patients the diagnosis of JE virus infection was made on the same day. Classical techniques for virus detection and antibody serology were also conducted in these patients. The indirect immunofluorescence for JE virus antigen detection in CSF cells proved to be a rapid and simple procedure for making a quick diagnosis of infection.

Mean survival time following intracerebral inoculation of dengue virus was reduced and the titre of the virus in the brain of immunosuppressed mice was markedly increased. A single dose of cyclophosphamide given 24 h after dengue virus i.c. or i.p. substantially reduced the number of antibody forming cells in the spleen. Three doses of dengue virus, each followed by cyclophosphamide 24 h later, produced specific hyporesponsiveness to the dengue virus but not to a heterologous virus (Coxsackie B4), with a reduction in antibody forming cells in the spleen of such animals against dengue virus but not against Coxsackie B4 virus. Adoptive immunity by antiserum was abolished along with increased titres of the virus in the brain of immunosuppressed mice but the protection could be restored by a second dose of antiserum. Pre-treatment of mice with immune or normal spleen cells i.v. or reconstitution of immunosuppressed mice by such cells had no effect. Thus, humoral antibodies play a crucially important role in host defence mechanism in recovery of mice from primary dengue virus infection.

The present study was undertaken to investigate if the suppressed cell-mediated immune responses observed in dengue type 2 virus (DV)-infected mice could be due to the cytotoxic factor (CF) produced in the spleens of DV-infected mice. We have observed that CF given intravenously (i.v.) kills splenic cells and reduces the total cells in the spleen. Mice treated with CF have a significantly depressed immune response to sheep erythrocytes, viz. delayed-type-hypersensitivity as measured by footpad swelling reaction at 24 hr; Jerne's antibody plaque-forming cells in the spleen; and migration inhibition of spleen cells in presence of antigen. These findings are similar to those seen earlier in DV-infected mice.

The immune response to Japanese encephalitis virus (JEV) was assessed in JEV-infected mice (mothers) and their offspring. The congenitally infected baby mice responded poorly in all assays for cell-mediated immunity. The total number of their splenic cells remained unaltered but the percentage of T cells was significantly reduced; a depressed delayed hypersensitivity response was seen against both homologous (JEV) and heterologous (sheep erythrocytes) antigens. In addition, significantly higher leukocyte migration inhibition (LMI) of spleen cells in the presence of specific antigen was observed. Adult mice infected during pregnancy demonstrated an impaired delayed hypersensitivity response to JEV antigen only. LMI was positive in mothers at 2 weeks post-partum, but not at later periods.

Changes in enzymes and metabolites of the carbohydrate metabolism in skeletal muscles were studied in mice after intracerebral inoculation of dengue type 2 virus. It was noted that lactic dehydrogenase, aldolase, phosphoglucoisomerase, phosphoglucomutase, GO-T and GP-T activity were enhanced initially by two- to three-fold, reaching a peak on day 5. As the illness appeared in mice, all the enzyme activities were lowered and were about three times less in the paralytic stage on the 8th day as compared to controls. Fructose-1,6-diphosphatase activity was increased on the 4th and 5th days but decreased later. Acid phosphatase increased abruptly from the 6th day while alkaline phosphatase activity was irregular. Creatine increased on the 4th and 5th days but diminished later. Glycogen decreased from the beginning and was lowest on the 5th day, but the levels increased later and were maximum in paralysed muscles. On the other hand, lactic acid began accumulating in the muscles and was maximum on the 5th day, then declined. Dengue virus was detected in the muscles from the 2nd day but higher titres were seen from the 6th day. Changes similar to the preparalytic stage of mice may occur in human beings, causing myalgia.

Transplacental transmission of Japanese encephalitis virus (JEV) has been demonstrated in consecutive pregnancies of mice. Pregnant mice inoculated intraperitoneally with JEV transmit the virus to the foetus. When such female mice were mated again after 6 months, the virus could be isolated from the foetuses of the ensuing pregnancy. The incidence of abortion was increased significantly though the neonatal deaths were considerably less than during the first pregnancy. Intra-uterine infection occurred in spite of the presence of HAI antibodies against JEV in the preconceptional sera of the mice. The findings of the present study indicate the value of such a system for further investigations of the pathogenesis of JEV infection during pregnancy in humans.

Our earlier observations indicate that adoptive transfer of spleen cells obtained from dengue type 2 virus (DV)-primed mice suppressed DV antigen-specific antibody secretion as detected by Jerne PFC technique. Findings of this paper indicate that the suppression was produced by non-glass-adherent cells, macrophage-depleted (by carbonyl iron) cells and by T lymphocytes of the spleen but not by the glass-adherent cells and B lymphocytes. The activity of these cells is dependent upon production of prostaglandin as shown by abrogation of their suppressor activity by pre-treatment of cells by indomethacin or aspirin which are known to block synthesis of prostaglandins.

We have observed that live macrophages play an obligatory role in transmission of dengue type 2 virus (DV)-induced suppressor signal to a subpopulation of T lymphocytes. The present study was undertaken to resolve whether transmission of the suppressor signal is mediated by a soluble factor or by direct cell-to-cell contact. It was observed that the suppressor factor (SF) remains adsorbed on the surface of macrophages and can be retrieved from them completely by DV-stimulated spleen cells kept in contact with them. Suppression of DV-specific IgM plaque-forming cells (PFC) in spleen-cell cultures does not occur when SF-adsorbed macrophages are separated from them by cell-impermeable membranes. Culture fluid of SF-adsorbed macrophages have no suppressor activity. The suppression of PFC occurs only when SF-adsorbed macrophages remain in contact with DV-stimulated spleen-cell cultures. Thus transmission of suppressor signal from SF-adsorbed macrophages to lymphocytes occurs only by physical contact of the plasma membranes of the interacting cells.